Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases or mixtures of enzymes are also often used.
To improve the cost and/or the performance of proteases there is an ongoing search for proteases with altered properties, such as increased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
The search for proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
One family of proteases, which are often used in detergents, are the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen R J and Leunissen J A M, 1997, Protein Science, 6, 501-523. One of these sub-groups is the Subtilisin family which includes subtilases such as BPN′, subtilisin 309 (SAVINASE®, NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41, Davail S et al. 1994, The Journal of Biological Chemistry, 269(26), 99. 17448-17453), subtilisin S39 (a subtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp. 1271-1279) and TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577).
The groupings indicated above were made based on primary sequence alignments, with only little consideration of three-dimensional structure. However, despite sequence homologies between subtilases belonging to the Subtilisin subgroup, modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase (such as the subtilisin BPN′ that was used by Siezen and Leunissen) may result in an incorrect three-dimensional model structure because of structural differences.
Recently the three-dimensional structure of subtilase TY145 have been elucidated and it was found that there are several differences between this and the three-dimensional structure of BPN′ also belonging to the Subtilisin subgroup of subtilases (PCT/DK2004/000066).
The differences between the three-dimensional structures of TY145 and BPN′ are confirmed by the three-dimensional structure of the subtilase “sphericase” from Bacillus sphaericus (PDB NO:1EA7, Protein Data Bank). The overall structure and many details of this subtilase are very homologous with the TY145 subtilase structure.
The subtilase JP170 and subtilases similar to JP170 are already known in the art, but the three-dimensional structure has not been disclosed for such subtilases.
The JP170 subtilase was described as protease A in WO 88/01293 to Novozymes. Later the patent application WO 98/56927 to Novozymes Biotech disclosed the amino acid (polypeptide) sequence of JP170 and the DNA sequence encoding JP170. In EP 204 342 the protease Ya was disclosed, and JP7-62152 and JP 4197182 to Lion Corp. disclosed the DNA sequence encoding protease Ya produced by Bacillus sp. Y that is homologous to JP170. In addition U.S. Pat. No. 6,376,227 to Kao Corp. discloses physical characteristics as well as DNA and polypeptide sequences of alkaline proteases KP43, KP1790 and KP9860 which are also homologous to JP170. Recently variants of the KP43, KP9860, SD-521 and Ya proteases among others were disclosed in EP 1209233. These proteases are highly homologous, and an alignment of KP43, KP9860, SD-521, Y and JP170 revealed at least 90% homology. Therefore JP170, Ya and SD-521 represent these proteases in the alignment of FIG. 1 of the present specification.
By 3D sequences is meant that the position of homologous residues are chosen by superposition of the 3D structures and subsequently the amino acid sequences are aligned based on these homologous positions.